Physical interaction of RECQ5 helicase with RAD51 facilitates its anti-recombinase activity

Abstract

Homologous recombination (HR) provides an efficient mechanism for error-free repair of DNA double-strand breaks (DSBs). However, HR can be also harmful as inappropriate or untimely HR events can give rise to lethal recombination intermediates and chromosome rearrangements. A critical step of HR is the formation of a RAD51 filament on single-stranded (ss)DNA, which mediates the invasion of a homologous DNA molecule. In mammalian cells, several DNA helicases have been implicated in the regulation of this process. RECQ5, a member of the RecQ family of DNA helicases, interacts physically with the RAD51 recombinase and disrupts RAD51 presynaptic filaments in a reaction dependent on ATP hydrolysis. Here, we have precisely mapped the RAD51-interacting domain of RECQ5 and generated mutants that fail to interact with RAD51. We show that although these mutants retain normal ATPase activity, they are impaired in their ability to displace RAD51 from ssDNA. Moreover, we show that ablation of RECQ5-RAD51 complex formation by a point mutation alleviates the inhibitory effect of RECQ5 on HR-mediated DSB repair. These findings provide support for the proposal that interaction with RAD51 is critical for the anti-recombinase attribute of RECQ5.

FIGURE 1.

Mapping RAD51-interacting domain of RECQ5. A, domain organization of human RECQ5 and schemes of RECQ5 deletion variants used in this study. RQC, RecQ C-terminal domain, which contains a Zn²⁺-binding motif that is essential for the helicase activity of RECQ5; PIM, PCNA-interacting motif. The dashed lines indicate location of the RAD51-interacting domain. B and C, CBD pull-down assay. Indicated RECQ5 variants were produced in E. coli as fusion with CBD and bound to chitin beads as described under “Experimental Procedures.” Beads were incubated with 293T cell extract (600 μg of protein), and RAD51 binding was analyzed by Western blotting using anti-RAD51 antibody. Blots were also stained with Ponceau S to visualize RECQ5 and its variants.

FIGURE 2.

Identification of amino acid residues of RECQ5 that are critical for RECQ5-RAD51 complex formation. Single alanine substitutions were created at the charged and aromatic residues in the region of RECQ5 spanning amino acids 654–674 that was found to be essential for interaction with RAD51. A, CBD pull-down assay was performed with the indicated RECQ5 mutants expressed in E. coli as fusion with CBD. Chitin beads coated with wild-type or mutant forms of RECQ5 were incubated with 293T cell extract (600 μg of protein) as described under “Experimental Procedures.” RAD51 binding was detected by Western blotting using anti-RAD51 antibody (bottom panel). RECQ5 proteins were visualized by Ponceau S staining (top panel). B, wild-type and mutant forms of RECQ5 were expressed ectopically in 293T cells as N-terminal fusions with a (His)₆-Xpress epitope tag. Extracts from these cells (800 μg of protein) were incubated with Ni-NTA-beads and bound proteins were analyzed by Western blotting using anti-RAD51 and Omni-probe antibodies. The latter antibody recognizes the (His)₆-Xpress tag.

FIGURE 3.

Role of the RECQ5-RAD51 complex in RECQ5-mediated inhibition of D-loop reaction. A, D-loop reaction scheme. B and C, effect of wild-type and mutant forms of RECQ5 on RAD51-mediated D-loop formation. RAD51^K133R (1 μm) was incubated with a 5′-end-labeled 90-mer oligonucleotide (3 μm nucleotides) to form a presynaptic filament, which was then incubated for 4 min with the indicated concentrations of wild-type or mutant forms of RECQ5 in the presence of RPA (135 nm), followed by addition of Hop2-Mnd1 (300 nm) and pBluescript form I DNA (50 μm base pairs). After a 6-min incubation, the reaction products were resolved in 0.9% agarose gels and visualized by phosphorimaging (left panels). Gels were quantified using ImageQuant software, and the concentration of D-loop products calculated as a percentage of the amount of product generated in the standard (Std) reaction carried out in the absence of RECQ5. The average values ± S.E. from three or more independent experiments are plotted (right panels). Asterisks denote statistically significant difference as compared with full-length (FL) RECQ5 by an unpaired Student's t test: *, p < 0.05; **, p < 0.005.

FIGURE 4.

Role of RECQ5-RAD51 complex in RECQ5-catalyzed displacement of RAD51 from ssDNA. A, scheme of DNA topology modification assay. RAD51 filaments pre-assembled on circular ssDNA are incubated with RECQ5. A relaxed plasmid DNA (dsDNA) is subsequently added to this reaction to trap RAD51 molecules displaced from ssDNA. RAD51 binding to topologically relaxed dsDNA induces lengthening of the DNA that can be monitored as a reduction in the DNA linking number upon treatment with eukaryotic DNA topoisomerase I. PK, proteinase K. B–D, effect of wild-type and mutant forms of RECQ5 on the stability of RAD51^K133R presynaptic filament. RAD51^K133R was assembled on M13 ssDNA (9 μm nucleotides) in the presence of 2 mm ATP and ATP-regenerating system, and then incubated with wild-type or mutant forms of RECQ5 (160 nm in B and 40, 80, and 160 nm in C and D) and RPA (150 nm) for 6 min before addition of relaxed DNA (7 μm bp) and wheat germ DNA topoisomerase I. Reactions were analyzed by electrophoresis in 1% agarose gel followed by ethidium bromide staining. Gels were quantified using ImageQuant software and the concentration of supercoiled DNA products calculated as a percentage of the amount of product generated in the reaction carried out in the absence of ssDNA, RECQ5, and RPA (lane 3). The tables under each gel show the average values from two or more independent experiments.

FIGURE 5.

Role of physical interaction between RECQ5 and RAD51 in the suppression of HR-mediated DSB repair in human cells. A, diagram of the DR-GFP reporter along with the homology-directed repair (HDR) product that gives rise to GFP+ cells. B, effect of overexpression of wild-type (WT) RECQ5 and the F666A mutant on HR repair of an I-SceI-induced DSB in HEK293 cells. Cells were transfected with an I-SceI expression vector along with the indicated amounts of either an expression vector for RECQ5 (WT or F666A) or empty vector (EV). Shown are the levels of repair relative to the mean value of a parallel set of EV transfections. Data represent the mean values of three independent transfections. Error bars reflect the S.D. Asterisks denote statistically significant difference as compared with WT RECQ5 by unpaired Student's t test (p = 0.0086 for 0.4 μg of plasmid DNA; p = 0.006 for 0.8 μg of plasmid DNA).